TRIM72-mediated degradation of the short form of p62/SQSTM1 rheostatically controls selective autophagy in human cells

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P62S, which contained the in-frame fusion of the GAL4 DNA binding domain. Yeast two-hybrid (Y2H) screening was performed by transforming a yeast strain (Mav203 strain) that contained the bait vector, with the pACT2 prey vectors for the human E3 cDNA expression library. Yeast transformants were first grown on a plate containing SD-2 (deficient in Leu and Trp) for selection of yeast cells containing both bait and prey vectors, and then transferred to SD-4 (deficient in Leu, Trp, His, and Ura) plates to screen for E3 ligase proteins that potentially interacted with human p62S. The interaction was confirmed by transforming yeast Mav203 cells with the indicated bait and prey vectors, and allowing the transformants to grow on the SD-2 or SD-4 agar plates for approximately 3 days at 30℃. Images of the colonies on both plates were recorded.

Co-immunoprecipitation (Co-IP), immunoprecipitation (IP) and immunoblotting (IB)
For Co-IP, HeLa cells transfected with plasmids were lysed in 800 μl Co-IP buffer (50 mmol/L Tris-HCl, 150 mmol/L NaCl, 5 mmol/L EDTA, and 1% NP-40, pH 7.4) supplemented with a protease inhibitor cocktail (Roche, Indianapolis, IN, USA). Cell lysates were centrifuged at 4℃ using 13,000 × g for 10 min, incubated with anti-Flag immunomagnetic beads (L-1011; Biolinkedin Biotech, Beijing, China) overnight at 4℃, and washed three times with Co-IP buffer. For immunoprecipitation, HeLa cells transfected with plasmids were lysed in 1 ml of IP buffer (50 mmol/L Tris-HCl, 150 mmol/L NaCl, 5 mmol/L EDTA, 0.1% SDS, and 1% NP-40, pH 7.5) supplemented with a protease inhibitor cocktail, and the cell lysates were centrifuged at 4˚C using 15,000 × g for 10 min, incubated with anti-Flag affinity gels (L-1013, Biolinkedin Biotech) overnight at 4℃, then washed three times with IP buffer. The immunoprecipitates from Co-IP and IP were denatured at 100℃ for 10 min in 2× SDS-PAGE loading buffer. The inputs, immunoprecipitates, and other cell lysates were subjected to SDS-PAGE, and then transferred to a polyvinylidene difluoride (PVDF) membrane (Bio-Rad, Hercules, CA, USA). The membranes were blocked with 10% nonfat milk at room temperature for 1 h, then incubated with the appropriate at room temperature for 1 h, washed three times with TBST, and the signals visualized by enhanced chemiluminescence (180-5001; Tanon Science and Technology, Shanghai, China) and detected by exposure to X-ray film, or detected by a 5200 Imaging System (Tanon). The density of bands was calculated using ImageJ software, version 1.8.0 (National Institutes of Health, Bethesda, MD, USA) when needed.

Expression and purification of recombinant proteins
The pGEX4T-1-GST-p62S, pGEX4T-1-GST-p62L, pET22b-TRIM72-His6, pET22b-LC3-His6, and pET22b-CYP26A1-His6 plasmids were expressed in BL21 Escherichia coli, then single cells were picked, cultured in 37ºC in 2 ml LB medium (10 g/L Tryptone, 10 g/L yeast extract, and 10 g/L NaCl) with respective resistance overnight, then the bacteria were inoculated into 500 ml LB medium and cultured for 6 h at 37℃, followed by incubation with isopropyl-β-dthiogalactopyranoside (Sangon, Shanghai, China) induction at 16℃ overnight. The next day, bacterial cells were centrifuged and lysated in phosphate-buffered saline (PBS), incubated with glutathione or Ni 2+ TA beads (GE Healthcare, Indianapolis, IN, USA) to enrich the respective proteins, followed by elution with 50 mmol/L reduced L-glutathione or with 1 mol/L imidazole dissolved in PBS. The eluted products were dialyzed in PBS supplemented with 15% glycerol prior to being aliquoted and preserved at -80℃.

Salmonella infection assay
An overnight culture of Salmonella strain SL1344 was diluted 1:25 and bacteria were grown at 37℃ until reaching OD600 at an absorbance of 1.0 − 1.2. HeLa cells were transfected with plasmids containing p62S, p62L, TRIM72, or its mutants for 24 h, treated with or without Rapamycin (2 μmol/L), BTZ (1 μmol/L), or BAF (20 nmol/L) for 1 h, and washed twice with PBS. The infection was conducted in antibiotic-free medium at a multiplicity of infection of 100. Salmonella were allowed to invade cells for 30 min, and then washed three times with PBS. The cells were then lysed and subjected to plate assays, and the Salmonella colony numbers were counted and calculated. The experiment was repeated three times.

Detection of cellular oxidatively damaged cellular proteins
HeLa cells were transfected with plasmids containing p62S, TRIM72, or its mutants for 24 h, and treated with or without Rapamycin (2 μmol/L) for 12 h, and BTZ (1 μmol/L), or BAF (20 nmol/L) for 1 h. Protein oxidation was determined using the OxyBlot Protein Oxidation Detection Kit (Millipore) according to the manufacturer's instruction as previously described [2,3]. Briefly, cell lysates were incubated for 20 min with 12% SDS, derivatized with DNP, stopped by Oxyblot Neutralization solution, and 3 μg of total proteins for each sample were resolved in SDS-PAGE, followed by transfer to nitrocellulose membranes. The membranes were blocked with the blocking buffer for 45 min at room temperature before incubation with antibodies against DNP (1:2000, D9656; Sigma-Aldrich) for 2 h, and subsequently with goat anti-rabbit horseradish peroxidasecoupled secondary antibodies (1:5000, ProteinTech) for 1 h at room temperature. Finally, the signals were visualized by enhanced chemiluminescence detected using a 5200 Imaging System (Tanon).

Statistical analysis
Data were analyzed using a two-tailed unpaired t-test or one-way analysis of variance with a Bonferroni post-hoc test using Prism 5 software (GraphPad Software, San Diego, CA USA). * P < 0.05 was considered to be significantly different. were used for the amplification of p62S cDNA. c Protein levels of p62L and p62S were detected in human HeLa cells and various mouse tissues by immunoblotting analysis, and no p62S expression was found. d shRNAs were designed to only target p62S but not p62L, to confirm the expression of p62S. Total mRNA extracted from HeLa cells stably transfected with p62S scramble or shRNA was used to measure the amount of p62L and p62S transcripts relative to GAPDH by quantitative PCR, and cell lysates were also subjected to immunoblot analysis with the indicated antibodies. Data are presented as the mean ± SD, and analyzed using the two-tailed unpaired t-test. ** P < 0.01, three independent experiments. ns non-significant and His6-tagged LC3. GST was a negative control. c Human p62S was required for autophagy activated by TRIM72. The p62 -/-MEF cells were transiently co-transfected with TRIM72 and p62L or p62S, and subjected to immunoblotting using the indicated antibodies 48 h later. The levels of lipidated LC3 were quantitated after normalization of the control as 1.00. TRIM72 tripartite motifcontaining 72